The Impact of CO2 Induced Anaerobiosis and Cold Shock on the
Flocculation Potential of Brewing Yeast
By: Francine Upshon
Supervisor: Professor Katherine Smart
INTRODUCTION
The basic role of yeast is to cause the fermentation of barley grains, which in turn
produces beer. Fermentation is the anaerobic conversion of fermentable sugars,
mainly maltose and glucose, by yeast, into ethanol, carbon dioxide and the
characteristic aroma compounds found in beer.
Figure 1. A small modern brewing
plant.
Wikimedia Commons GNU Free
Documention Licence
The behaviour of yeast cells during fermentation, especially the clumping or
flocculation of yeast cells, is extremely important for the brewing industry
because they can affect the final flavour, texture, alcohol level and clarification of
the beer (Jibiki et al., 2001). Top fermenting yeasts tend to produce a light frothy
‘head’ on the beer and are used to produce ales. Bottom fermenting yeasts are
used to produce lagers which tend to retain more ‘fizz’. There are many natural
by-products of fermentation such as diacetyl and acetaldehyde. These natural byproducts all contribute to the different aroma compounds and flavours found in
certain types of beer.
The ideal flocculation percentage for brewers would be as close to 100% as
possible, as that would signify complete cell flocculation. A flocculation
percentage of 0 on the other hand would indicate that the yeast is fully dispersed
throughout the beer, resulting in a cloudy product with a texture that is
undesirable for consumers.
Production scale brewery fermentations are traditionally carried out at around
15oC under anaerobic conditions. This is considerably lower than the 25oC
temperature used to quantify flocculation in a laboratory which is measured under
aerobic conditions. Consequently, these laboratory conditions may not give a true
representation of the conditions that the yeast are subjected to during production
scale brewery fermentations. Hence, it may be more appropriate to analyse
flocculation performance at a temperature similar to that found during production
scale brewery fermentations.
The aim of this study was to investigate the impact of anaerobic conditions and
temperature on the flocculation potential of brewing yeast.
Fermentation was carried out at three different temperatures conditions: 4oC to
represent cold storage, 15oC as the temperature at which fermentation is
commercially performed and 25oC as the standard laboratory temperature.
Brewing Yeast
A brewing yeast cell goes through 4 stages: a lag phase, a growth
phase(reproduction), a fermentation phase and a sedimentation phase. Cells are
activated from dormancy when they are added (or pitched) to the wort. Once
sufficient food reserves are built up, the yeast cells are then able to reproduce by
budding, and fermentation commences. During fermentation the yeast is in
suspension in the wort from 5-7 days. After this time, the yeast cells begin to
flocculate and sediment.
Figure 2. These are budding cells of the brewers yeast,
Saccharomyces cerevisiae. Scars form where daughter cells have
broken away from the parent cell. This unicellular fungus ferments
sugars into alcohol. Gordon Beakes © University of Newcastle
upon Tyne. Image courtesy Centre for Bioscience, the Higher
Education Academy, ImageBank
http://www.bioscience.heacademy.ac.uk/imagebank/.
Brewing yeast can be divided into two main types; ale and lager. These yeasts
share many similarities, but they also have different electrical properties,
hydrophobicities and surface chemical compositions (Dengis et al., 1995). The
most marked difference between the two yeast strains however, is observed
towards the end of fermentation and is due to the expression of cell surface
proteins.
Saccharomyces cerevisiae (S. cerevisiae) is an ale yeast species. Four strains of
this yeast were used in this study. Ale strains are generally more hydrophobic
than lager strains, due to a higher concentration of surface proteins containing
protonated amino and carboxylate groups. Ale fermentations are traditionally
carried out over 5-6 days at between 12-18oC where the yeast cells rise to the
top of the fermenter due to their affinity for CO2 bubbles (Ritcey, 1997). Ale
strains are given the phenotypic description NewFLO (Stratford and Assinder,
1991). The NewFLO phenotype strain is characterised by flocculation that is
inhibited by mannose, maltose, glucose or sucrose.
Flocculation can be defined as the “phenomenon wherein yeast cells adhere in
clumps and either sediment rapidly from the medium in which they are
suspended or rise to the surface” (Stewart and Russell, 1981). The most widely
accepted hypothesis to explain the mechanism of flocculation is the lectin-like
theory of flocculation (Miki, 1982). This hypothesis suggests that specific lectinlike cell surface proteins known as zymolectins, selectively bind to proteins
containing mannose polysaccharides (mannoproteins) present on the walls of
neighbouring yeast cells (See Fig. 3).
Lectin
Figure 3. In the lectin-like theory of
flocculation, lectins appear at the cell surface
in the stationary phase and specifically bind
to mannan carbohydrates of adjacent yeast
cells (after Touhami et al, 2003).
Cell
Mannan
Flocculation is influenced by the properties of the yeast cell wall and the nature of
the culture medium or wort. It is important that strain variability is taken into
account when considering culture temperature and composition as individual
yeast strains respond differently to environmental conditions. In addition to
temperature, the concentrations of ethanol, trace elements and especially
dissolved oxygen can all affect flocculation. Anaerobic conditions lead to the
differential expression of cell wall mannoproteins (Smart, 2003). It is presumed
that this change in expression alters cell wall composition and structure,
favouring lectin-receptor interactions (Smart, 2003).
METHODS
Ale yeast strains were cultured at a range of temperatures both aerobically and
anaerobically and their ability to flocculate determined.
The results from two ale yeast strains are reported here (SCB7, SCB8, Scottish
Courage Brewing Ltd, Technical Centre, Edinburgh, UK.) Each strain was grown
aerobically on a Yeast Peptone Dextrose (YPD) plate at 25oC, then a starter
culture produced by inoculating a single colony from each growth plate into
100ml of YPD in a 250ml sterile flask. The flasks were then put in an orbital
shaker at 120 rpm at 25oC for 48 hours and the cell concentration determined by
counting the cells under a microscope on a special slide called a haemocytometer
(haemocytometers have a calibrated grid commonly used for counting blood cells,
hence the name). A volume of this cell suspension was then inoculated into
100ml of YPD in a 250ml sterile flask to obtain a final cell concentration of 1x106
cells/ml.
The yeast cells were then incubated for 48, 72 and 96 hours in YPD at 25oC, 15oC
or 4oC, shaking or static, in either aerobic or CO2 induced anaerobic conditions.
The choice of 4oC was intended to represent cold storage, 15oC as the
temperature at which fermentation is traditionally carried out and 25oC as the
standard laboratory temperature. Anaerobic incubation was achieved using an
Oxoid Anaerogen® pack and a 3.5L gas jar.
Cell populations were harvested by centrifugation and the flocculation potential
measured.
The sedimentation rate, which is dependent upon the viscosity of the medium,
floc size and mass is the standard method of analysis (Stratford, 1992). A major
commercially used test of this type is called the Helm’s test. This method was
used in this study. The optical density (OD) of a yeast solution is measured over
a set time period. The quantification of cell sedimentation therefore gives an
indication of the physical behaviour of the strain in liquid culture, and the rate
and degree to which it flocculates.
Data was analysed by comparing the % flocculation against the fermentation time
at different temperatures under aerobic or anaerobic conditions. Fisher’s
protected least significant difference test (Fisher’s PLSD) was used to determine
significant differences between the groups.
RESULTS
For populations of the SCB7 ale yeast strain, when incubated aerobically at 25oC
there was a significant increase in flocculation potential, however when incubated
anaerobically at 25oC, there was a significant decrease in flocculation potential.
For SCB8, under both aerobic and anaerobic conditions at 25oC, the flocculation
potential was maintained throughout the time period assessed at approximately
the same value that was achieved after 48 hours, thus there was no significant
change.
At 15oC, the flocculation potential of SCB7 progressively decreased under both
aerobic and anaerobic conditions. With SCB8, the flocculation potential peaked at
the 72 hour time point under both gaseous conditions, but there was no further
increase in the flocculation potential with any further incubation.
For SCB7 under aerobic incubation at 4oC there was a progressive increase in
flocculation potential from 72 to 96 hours. Under anaerobic incubation at 4oC,
flocculation progressively increased. Aerobic and anaerobic incubation of SCB8 at
4oC lead to a progressive increase in the flocculation potentials achieved under
both gaseous conditions.
DISCUSSION
The yeast cell wall is a dynamic structure that can adapt in response to many
physiological and morphological changes. The composition of the cell wall
changes when it is exposed to different growth conditions: including temperature,
pH, aeration, the mode of cultivation and the available carbon source.
The Helm’s test is currently performed with incubation under aerobic conditions at
25oC, whereas commercial brewing occurs under anaerobic conditions at 15 oC.
Lawrence and Smart (in press) investigated the impact of CO2 induced
anaerobiosis on flocculation performance and found that in all yeast strains
assessed, the rate of onset and extent of expression of flocculation was modified
in response to this changing gaseous condition. They proposed that the
quantification of flocculation during CO2 induced anaerobiosis gave a more
representative and predictive assessment of production scale brewery
fermentation than the current method performed under aerobic conditions.
Aerobic Incubation of Ale Yeast Strains at 250C, 150C and 40C
Under aerobic incubation at 25oC, 15oC and 4oC, both strains demonstrated a
flocculation profile different to the other. This may have been because the
reduction in temperature had opposing effects on each strain, either inducing or
repressing the formation of a cell wall component involved in flocculation. The
flocculation response to incubation under different temperature conditions is
therefore presumed to be strain specific.
Anaerobic Incubation of Ale Yeast Strains at 25oC, 15oC and 4oC
At 25oC, the yeast cells were shaken during incubation. At 15oC and 4oC however,
the yeast cells remained static. Agitation can have two opposing effects
depending on how vigorous the yeast cells are shaken. A higher flocculation rate
can occur as a result of enhanced particle collision rates, but depending on the
100
90
% Flocculation
80
70
60
SCB7
50
SCB8
40
30
20
10
0
48
72
96
Time (hours)
a) 25oC
100
90
% Flocculation
80
70
60
50
40
SCB7
30
SCB8
20
10
0
48
72
96
Time (hours)
% Flocculation
b) 15oC
100
90
80
70
60
50
40
30
20
10
0
SCB7
SCB8
48
72
96
Time (hours)
o
c) 4 C
Figure 4. Flocculation potential of ale brewing yeast strains in aerobic conditions at 25oC,
15oC and 4oC. Yeast populations were grown in aerobic conditions in YPD, for 48, 72 and 96
hours. Cells were harvested by centrifugation and flocculation was measured using a
modified version of the Helm’s test. The data represents the mean of triplicate analysis.
Error bars represent the standard error.
% Flocculation
100
90
80
70
60
50
40
30
20
10
0
SCB7
SCB8
48
72
96
Time (hours)
o
% Flocculation
a) 25 C
100
90
80
70
60
50
40
30
20
10
0
SCB7
SCB8
48
72
96
Time (hours)
o
% Flocculation
b) 15 C
100
90
80
70
60
50
40
30
20
10
0
SCB7
SCB8
48
72
96
Time (hours)
c) 4oC
Figure 5. Flocculation potential of ale brewing yeast strains in anaerobic conditions. Yeast
populations were grown in YPD at 25oC, for 48, 72 and 96 hours. Anaerobic conditions were
achieved by incubation in an anaerobic gas jar containing an Oxoid Anaerogen® pack
inside. The data represents the mean of triplicate analysis. Error bars represent the
standard error.
strength of the agitation, it can also cause particle breakage, leading to a
decrease in floc size and a slower rate of sedimentation (Domingues et al.,
2000). Thus, agitation may have had an affect on the flocculation potentials
achieved under each temperature condition.
At all temperature conditions, SCB8 achieved a higher flocculation potential under
both aerobiosis and CO2 induced anaerobiosis than SCB7, indicating that SCB8
may be more stress tolerant than SCB7. Both strains achieved their highest
flocculation potentials under aerobic incubation at 25oC. There was no pattern
between the flocculation profiles of SCB7 and SCB8 observed under incubation at
each different temperature. This response is likely to be the consequence of a
strain specific effect.
Previous research carried out by Lawrence and Smart (unpublished data), has
shown that the rate of onset and extent of expression of flocculation of two
different lager yeasts, SCB2 and SCB3 also appears to be strain dependant. The
flocculation profile of these lager yeasts when incubated under aerobic conditions,
completely differed from those seen under incubation in CO2 induced anaerobic
conditions. Therefore the results from this study are in agreement with the
observations of other laboratories. These studies suggest that it may be
necessary to incorporate temperature as well as CO2 induced anaerobiosis when
analysing the flocculation performance of yeast, to better represent the conditions
found during production scale brewery fermentations.
CONCLUSION
The flocculation potential of ale yeast strains was indeed influenced by CO2
induced anaerobiosis and temperature, although the rate of onset and extent of
expression of flocculation appeared to be strain dependant.
These findings indicate that it may be necessary to consider temperature as well
as CO2 induced anaerobiosis when analysing the flocculation performance of yeast
strains under laboratory conditions. This would give a better representation of the
conditions found during production scale brewery fermentations, so that a more
accurate and predictive assessment of the performance of these attributes on
flocculation potential can be made.
REFERENCES
Dengis, P. B, Nelissen, L. R and Rouxhet, P. G (1995) Mechanisms of Yeast
Flocculation: Comparison of Top and Bottom-Fermenting Strains, Applied and
Environmental Microbiology, 61, 718-728.
Domingues, L, Vicente, A. A, Lima, N and Teixeira, J. A (2000) Applications of
Yeast Flocculation in Biotechnological Processes, Biotechnology and Bioprocess
Engineering, 5, 288-305.
Jibiki, M, Ishibiki, T, Yuuki, T and Kagami, N (2001) Application of Polymerase
Chain Reaction to Determine the Flocculation Properties of Brewer’s Lager Yeast,
Journal of the American Society of Brewing Chemists, 59, 107-110.
Miki, B.L.A, Poon, N.H, James, A.P and Seligy, V.L (1982) Possible mechanism for
flocculation interactions governed by gene FLO1 in Saccharomyces cerevisiae.
Journal of Bacteriology, 150, 878–889.
Ritcey, L. L (1997) Cell Wall Properties Affecting Brewing Yeast Flocculation.
Thesis (MSc) University of Nova Scotia.
Smart, K. A (2000) Brewing Yeast Fermentation Performance, Blackwell Science,
pp 78, 80, 84, 119, 121.
Stratford, M and Assinder S (1991) Yeast Flocculation: Flo1 and NewFlo
Phenotypes and Receptor Structure, Yeast, 7, 559-574.
Stratford, M and Keenan, M. H. J (1987) Yeast Flocculation: Kinetics and Collision
Theory, Yeast, 3, 201-206.
Stewart, G. G and Russell, I (1981) Brewing Science, Vol. 2, Academic Press,
Chapter 2, pp 61-91.
Author Profile:
Francine Upshon: Fran is 20 years old and studied Applied Biology at the
University of Nottingham, based at the Sutton Bonington campus. She graduated
in 2007 with an upper second BSc with honours degree. Fran studied a wide
range of topics on her course including many plant, animal and microbial
modules. She was particularly interested in the microbiology of the brewing
process and is currently applying for a job within the brewing industry.